Electron-microscopy of Fasciola hepatica. V. Peroxidase localization

EXPER-NTAL

PARASITOLOGY 23, 221-227

(1968)

ElectromMicroscopy Peroxidase

of Fasciola hepatica. Localization1

L. T. Threadgold Biology

Department, (Submitted

Rice

and

C. P. Read

University,

for publication,

V.

Houston, 10 April

Texas 77001 1968)

THREADGOLD, L. T., AND READ, C. P. 1968. Electron-microscopy of Fasciola hepatica. V. Peroxidase localization. Experimental Parasitology 23, 221-227. A peroxidase has been demonstrated histochemically in Fasciola hepatica. It is associated with the outer and inner limiting, and cristae membranes of the mitochondria of the tegument, muscle and parenchyma. Its presence has not been shown in the mitochondria of the intestine, vitelline glands or excretory system. INDEX DESCRIPTORS: Peroxidase; Fasciola hepatica; Electron microscopy; Enzymology; Mitochondria.

a Thermos flask. Flukes were sliced transversely in a fixative containing 3% glutaraldehyde in Millonig buffer, pH 7.2, PIUS 0.5 mM calcium chloride and 3% sucrose and immediately transferred to fresh fixative for 2 hours at 4°C. The tissue was washed overnight in Millonig buffer, pH 7.2, containing 3% sucrose at 4°C and then divided up and treated in the following ways:

Pennoit de Cooman and van Grembergen (1942) reported reactions for catalass and peroxidase in homogenates of Fasciolu hepatica. They concluded, however, that the peroxidase reaction was a pseudoenzymatic one, being due to the blood ingested from the host. Recently, Threadgold et al. (in press) showed by the diamino-benzidine hydrochloride (DAB) method and electron-microscopy that the tapeworm Hymerwlepis &&a contained a true peroxidase. The enzyme was confined to the mitochondria of the tegument and parenchyma, and was not detected in muscle mitochondria. Because of the above it was decided to apply the DAB technique and electronmicroscopy in determining whether a peroxidase was present in Fasciola. MATERIALS

AND METHODS

Adult Fasciola hepatica were obtained from cattle livers and brought to the laboratory in Hedon-Fleig’s medium, containing 2000 units/ml of penicillin G. and 0.1 mg/ml streptomycin and kept at 37°C in 1 This work was supported by the National Institutes of Health, Health Service (AI 01384).

a grant from U. S. Public

1. Test mediuma 10 ml of 0.05 M TrisHCl buffer, pH 7.2 containing 5 mg diamino-benzidine hydrochloride and 0.1 ml of freshly prepared 1X hydragen peroxide. Incubation 20 minutes. 2 Control medium. The test medium ’ without hydrogen peroxide. Incubation 20 minutes. 3. Inhibition medium. Preincubation for 30 minutes in either 1.0, 0.1, or 0.01 mM potassium cyanide in 0.05 M Tris-l%Cl buffer, pH 7.2, followed by incubation for 20 minutes in the test medium plus potassium cyanide at the same molarity as the particular preincubation. 4. Reactivation medium. After 30 min‘Dr.

221

C. W. Philpott,

personal

communication.

THRFADGOLD AND READ

222

KEY TO CAPTIONS: E = Excretory muscle

cell;

P = Parenchymal

cell;

epithelial cell; T = Tegumentary

I = Interstitial material; cell; V = Vitelline cell.

M = Mitochondria

PEROXIDASE

LOCALIZATION

TABLE

IN

Fawiola

223

I Results

Observed by light microscopy

Treatment.

Observed by electron-microscopy

1. Test medium

**

Pa

2. Control.

-

N

-

h-

* or *

P

Test medium less HTO,

3. Inhibition.

Test medium Test medium Test medium

f f f

1.0 mM KCN 0.1 n&f KCN 0.01 m&f KCN

4. Reactivation.

Test medium after washing out KCN at 1.0, 0.1, or 0.01 m&f

5. Test medium

+ NaF 1.0 mM

-

N

6. Test medium 10 minut.es

after Hz0 at 90°C for

-

N

7. Test medium or 114 m&f

after ascorbic acid at 57 mM

-

n P = Positive

reaction;

N = Negative

reaction;

utes in either 1.0, 0.1, or 0.01 mM potassium cyanide in 0.05 M Tris-HCI buffer, pH 7.2, the tissue was washed for 1 hour in Millonig buffer, pH 7.2, plus 3% sucrose and then incubated in the test medium for 20 minutes. 5. Heat treatment. Tissue was pretreated for 10 minutes in distilled water at 90°C and then incubated in the test medium for 20 minutes. 6. Sodium fluoride inhibition. Preincubation for 30 minutes in 1.0 mM sodium fluoride in 0.05 Tris-HCl buffer, pH 7.2, and then incubated in test medium plus 1.0 mM sodium fluoride for 20 minutes. 7. Ascorbic acid inhibition. Preincuba-

** = Dark

brown;

* = Light

brown;

-

= White.

tion in 57 or 114 mM ascorbic acid in 0.05 M Tris-HCl buffer, pH 7.2, for 10 minutes and then incubation in the test medium for 20 minutes. Following these incubations, some of the tissue was observed by light microscopy and the remainder was post-osmicated for 90 minutes in 1X osmic acid in Millonig’s buffer containing 3% sucrose. The tissue was then dehydrated and embedded in Epon. Silver sections were cut on a PorterBlum MT 2 and viewed unstained or double stained with alcoholic uranyl acetate and lead citrate (Reynolds, 1963). Sections were observed in an RCA EMU 3F and photographs taken at x11-22,000 magnification.

FIG. 1. Test medium. Tegumentary mitochondria. A positive reaction is shown by the marked density on and between (arrow) the membranes. Tissue post-osmicated: section unstained. X45,000. FIG. 2. NaF inhibited. Tegumentary mitochondria. No reaction product is evident on or between membranes. Tissue post-osmicated: section unstained. X35,000. FIG. 3. KCN inhibited. Tegumentary mitochondria. No reaction product is evident on or between membranes. Tissue post-osmicated: section &stained. X40,000. FIG. 4. Reactivated system after KCN inhibition. Tegumentary mitochondria. The membranes show increased electron density (arrow) compared with Fig. 3. Tissue post-osmicated: section unstained. x45,000.

224

THREADGOLD

AND

REAkI

FIG. 5. Test medium. The mitochondria of muscle (M) and parenchyma (P) are positive, but those of the tegumentary cell (T) show no activity. Tissue post-osmicated: section unstained. X37,500.

PEROXIDASE

LOCALIZATION

OBSERVATIONS

Table I presents the results of incubating tissue in the various substrates and inhibiting media. The results show that a true peroxidase is present. observations The electron-microscope show that the reaction product is confined to mitochondria. The reaction is strongest in the mitochondria of muscle (Figs. 5, 6, 8, 9), and progressively less strong in those of the surface syncytium of the tegument (Fig. l), the parenchyma (Figs. 5, 7, 9), and the tegumentary cells, although the latter in some cases did not show any activity (Fig. 5). No reaction was present in the cells of the intestine (Fig. 8), excretory system ( Fig. 7), including the flame cells, or vitelhne glands (Fig. 6) even when mitochondria of adjacent parenchyma or muscle were positive. In unstained sections the reaction appeared as an increased electron density of the outer, inner, and cristae membranes of the mitochondrion (Figs. 1, 9, 12). At high magnification this density was resolved into discrete granules or groups of granules on or attached to the membranes (Fig. 12). In strong reactions, the deposits occluded the space between outer and inner limiting membranes and the intracristal space (Fig. 12). No reaction was present in the mitochondria of any tissue after being incubated in media without substrate or in test media including potassium cyanide (Fig. 3). In tissue washed free of potassium cyanide, the peroxidase was reactivated in ah tissues in which it had previously been shown to be present, although the reaction

225

IN f;‘asCioh

was reduced in intensity (Fig. 4). action occurred in tissue incubated medium after preincubation in hot sodium fluoride (Figs. 2, 10, ll), corbic acid.

No rein test water, or as-

DISCUSSION

The observations of this study, especially the reactivation of the system after potassium cyanide inhibition and irreversible inhibition after sodium fluoride and heat treatment, provide strong evidence that the reaction is enzymatic. The inhibition following ascorbic acid treatment suggests that the enzyme is a peroxidase, rather than a catalase. Kovacs and Mazarean (1965) have shown that in Staphylococcus cultures ascorbic acid reduces the oxygen tension of the medium; peroxidase is inhibited by low oxygen tension, but catalase activity is stimulated. These results and the locahzation of the reaction deposits within mitochondria indicate that a true peroxidase is present in Fa.scioZu. The conclusion of Pennoit de Cooman and van Grembergen (1942) that peroxidase activity is due to ingested haemoglobin, and therefore only pseudoenzymatic, is incorrect. Threadgold et al. (in press) showed that the peroxidase in Hymenolepti diminuta was confined to the mitochondria of the tegument and parenchyma. The results for FascioZu, therefore, differ from those in Hymenobpis in certain respects. In Fasciola, the outer membrane of mitochondria is reactive, whereas it is not in muscle mitochondria give Hymenolepis; the strongest reaction in Fasciola but are

FIG. 6. Test medium. The mitochondrion of the muscle (M) is positive but the mitochondria of the juvenile vitelline cell (V) are negative. Tissue post-osmicated: section unstained. X22,000. FIG. 7. Test medium. The parenchymal mitochondria (P) are positive but those of the excretory epithelium (E) are without reaction product. Tissue post-osmicated: section unstained. X27,500. FIG. 8. Test medium. The muscle mitochondria (M) show a positive reaction, which is absent from the mitochondria of the intestinal epithelium (I). Tissue post-osmicated: section stained. X33,000.

226

THIREADGOLD AND BEAD

of both the muscle and FIG. 9. Test medium. A positive reaction is present in the mitochondria parenchyma (arrows), but is noticeably more intense in the former. Tissue post-osmicated: section unstained. X66,000,

PEROXIDASE LOCALIZATION

negative in Hymmolepis. In general the peroxidase appears to be at a higher concentration and more cyanide-resistant in Fcmiolu than in Hymmwbpis, since it takes potassium cyanide at 0.1 mM concentration and water at 90°C for 10 minutes to inhibit the enzyme in the former but only at 0.01 mit4 and for 5 minutes in the latter. It is not possible at present to assign a specific role for peroxidase in Fasciolu. However, the current concept of enzyme distribution in mitochondria postulates that the enzymes of the respiratory chain are in or upon the membranes, whereas those of the Krebs or fatty acid cycle are probably in the matrix or very loosely attached to the membranes (Lehninger, 1965). The location of the peroxidase described here suggests that it may be related to respiration. Perhaps the function of the enzyme is not primarily the detoxification of hydrogen peroxide by reducing it to oxygen and water, but rather the catalysis of coupled oxidations by means of hydrogen peroxide formed in a primary oxidation, as has been suggested for catalase by Keilin and Hartree ( 1945).

227

m Fasciolu ACKNOWLFBGMENTS

This work was undertaken while one of us (L.T.T.) was a Visiting Lecturer in the Biology Department of Rice University, Houston, Texas. We thank Dr. C. W. Philpott for the use of his electron-microscope facilities, and Dr. H. Isseroff for supplying the adult Fa.scioZa heputica. REFERENCES LEHNINGER, A. L. 1965. The “Mitochondrion,” 263 pp. Benjamin, New York. KELIN, D., AND HARTREE, E. F. 1945. Properties of catalase. Catalysis of coupled oxidations of alcohols. Biochemical Journal 39, 293-301. KOVACS, E., AND MAZARFZAN, H. H. 1965. Investigation of the action mechanism and induction of catalase in the culture of Staphylococcu.s auwus. Enymologiu 30, 19-28. PENNOIT DE COOMAN, E., AND VAN GREIUBERGEN, G. 1942. Vergelijkend Onderzoek van het Fermentensystem bij vrij en parasitaire Plathelmithen. Kongelige Vlaamse Academic UM Wetenschuppen, Klassen der Wetenschappen. Verhandelingen, Antwerp, 4, 7-77. ( In Dutch. ) REYNOLDS, E. S. 1983. -The use of lead citrate at high pH as an electron-opaque stain in electron-microscopy. Journal of CeZl Biology 17, 208-212. THREADGOLD, L. T., ARME, C., AND READ, C. P. The ultrastructural localization of a peroxidase diminuta. in the tapeworm, Hymenolepis Jolbrd of Parasitol0gy. (In press.)

FIG. 10. KCN inhibited. Parenchymal cell mitochondria pare with Fig. 9. Tissue post-osmicated: section unstained. FIG. 11. NaF inhibited. The mitochondria of muscle ( M product. Tissue post-osmicated: section unstained. X26,000. FIG. 12. Test medium. Tegumentary mitochondria show to the membranes (arrows). Tissue post-osmicated: section

do not show any reaction deposits. ComX37,500. ) and parenchyma (P ) are without reaction granular reaction deposits unstained. X82,500.

on and attached